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Department of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [J. G., S. Ka., J. A. R., B. F.], and Department of Obstetrics and Gynecology, Kanazawa University, School of Medicine, Ishikawa 920-0934, Japan [M. T., S. Ky., M. I.]
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Telomerase is a specialized DNA polymerase responsible for the replication of chromosomal ends, or telomeres. Telomerase is highly active in immortalized cell lines and >85% of human cancers but is inactive in most somatic cells (5 , 6) . The enzyme is a ribonucleoprotein complex composed of an essential RNA template and several associated proteins, among which is the essential catalytic subunit named TERT. Recently, the hTERT gene has been cloned by several independent groups and it has been found to be expressed at high levels in primary tumors and cancer cell lines but repressed in most somatic tissues (7, 8, 9, 10) . Recent data suggest that hTERT is a key determinant of the telomerase activity. This includes the finding that hTERT expression is highly correlated with telomerase activity (7, 8, 9, 10) and that the ectopic expression of hTERT in telomerase-negative cells is sufficient to reconstitute telomerase activity (11, 12, 13) and extend the life span of normal human cells (14) . More recently, Hahn et al. (15) showed that ectopic expression of hTERT is required, but is not sufficient, for direct tumorigenic conversion of normal human epithelial and fibroblast cells.
The promoter region of the hTERT gene has also been cloned
recently (16, 17, 18)
. The promoter is highly G/C-rich
and lacks both TATA and CAAT boxes but contains binding sites for
several transcriptional factors, including Myc and Sp1. Deletion
analysis of the hTERT promoter identified a core promoter region of
200 bp upstream of the transcription start site. Transient
expression assays revealed that the core promoter is significantly
activated in cancer cell lines but is repressed in normal primary
cells.
Because the hTERT gene is highly active in tumor cells but repressed in most normal cells and because its expression is regulated at the transcription level, we hypothesized that the hTERT promoter may be used for tumor-specific expression of transgenes. In the present study, we have assessed the capability of adenovirus-mediated transgene expression induced by the hTERT promoter and the feasibility of targeting the pharmaceutical effects of the Bax gene, a strong proapoptotic gene, to tumors by this promoter both in vitro and in vivo.
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Materials and Methods |
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Analysis of in Vitro Gene Expression.
Human lung cancer cell lines A549 and H1299 and cervical cancer cell
line HeLa were originally obtained from American Type Culture
Collection and maintained in our laboratory (M. D. Anderson
Cancer Center). Human colon cancer cell lines DLD1 and LoVo were
obtained from Dr. T. Fujiwara (Okayama University, Okayama,
Japan). NHFB cells and NHBE cells were purchased from Clonetics
(San Diego, CA) and cultured in media recommended by the manufacturer.
Cells were plated 1 day prior to vector infection at densities of
1x105/well in a 24-well plate. Cells were then
infected with adenoviral vectors at a MOI of 1000 viral particles/cell.
Twenty-four h after infection, cells were either stained with X-Gal to
visualize ß-galactosidase expression or harvested for biochemical
analysis of ß-galactosidase activity.
Biochemical Analysis.
Cultured cells were lysed or tissues from BALB/c mice were homogenized
in ß-galactosidase assay buffer. Cell or tissue debris was then
removed by microcentrifugation. Protein concentrations were determined
using a kit from Pierce according to the manufacturer’s instructions.
ß-galactosidase activities were determined using a luminometer and a
Galacto-Light Chemiluminescent Assay kit from Tropix, Inc. (Bedford,
MA).
Cell Viability Assay.
Cells were plated on 96-well plates at 1 x 104 per well 1 day prior to virus infection.
Cells were then infected with adenoviral vectors at a total MOI of 1500
viral particles/cell. Cells were divided into four groups according to
the viral vector system given: Ad/CMV-GFP + Ad/PGK-GV16,
Ad/GT-Bax + Ad/CMV-GFP, Ad/GT-Bax + Ad/hTERT-GV16,
or Ad/GT-Bax + Ad/PGK-GV16. In each group, the ratio of the
two viral vectors was 2:1, a ratio shown to be optimal for the
induction of transgene expression in previous experiments
(20)
. PBS was used for mock infection. The cell viability
was determined by XTT assay using a Cell Proliferation Kit II (Roche
Molecular Biochemicals) according to the manufacturer’s protocol. In
each treatment group, quadruplicate wells were measured for cell
viability.
Apoptosis Analysis by Flow Cytometry.
Cells were plated at densities of 1 x 106/100-mm plate 1 day prior to infection. The
cells were then infected with recombinant adenoviral vectors at a MOI
of 1500 viral particles/cell. Forty-eight h later, both adherent and
floating cells were harvested by trypsinization, washed with PBS, and
fixed in 70% ethanol overnight. Cells were then stained with propidium
iodide for analysis of DNA content. Apoptotic cells were
quantified by flow cytometric analysis performed in the Flow Cytometry
Core Laboratory at our institution (M. D. Anderson Cancer
Center).
Animal Experiments.
All of the mice were cared for according to the Guide for the Care and
Use of Laboratory Animals (NIH publication number 85–23) and the
institutional guidelines of The University of Texas M.D. Anderson
Cancer Center. In vivo infusion of adenoviral vectors into
and subsequent tissue removal from BALB/c mice were done as described
previously (19)
. In the s.c. tumor model, 5 x 106 H1299 cells were inoculated s.c. into
the dorsal flank of 6- to 8-week-old nude mice (Harlan Sprague Dawley,
Indianapolis) to establish tumors. After tumors reached 5 mm in
diameter, mice were given three sequential intratumoral injections of
9 x 1010 viral particles in a
volume of 100 µl per dose. Tumor sizes were measured three times a
week. Tumor volumes were calculated using the formula
a x b2 x 0.5, where a and b represent the larger
and smaller diameters, respectively.
Histochemistry Study.
For H&E staining, sectioned tissues or tumors were processed by the
Histology Laboratory in the Department of Veterinary Medicine and
Surgery at our institution. For X-Gal staining, 8-µm-thick frozen
sections were fixed with 50% ethanol and 50% methanol for 20 min at
-20°C. The fixed sections were then stained with a solution,
containing 5 mM
K4Fe(CN)6, 5 mM
K3Fe(CN)6, 2 mM
MgCl2, and 1 mg/ml X-Gal, at 37°C overnight and
were finally counterstained with Nuclear Fast Red (Sigma).
Analysis of Serum AST and ALT.
Blood was drawn from the tail vein of mice 48 h after adenovirus
infusion. The levels of serum AST and ALT were measured as described
previously (21)
.
Statistical Analysis.
Differences among the treatment groups were assessed by ANOVA using
statistical software (StatSoft, Tulsa, OK). P 
0.05 was considered significant.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. CMV and hTERT promoter activity differed by only 2- to
10-fold in cancer cells compared with more than a 500-fold difference
in normal cells (Fig. 1B)
. In all of the cells tested, hTERT
promoter activity was significantly higher in cancer cells than in
normal cells (P 0.05). These results
together demonstrated that the hTERT promoter was highly active in a
variety of cancer cell lines but not in normal cells, thus suggesting
that the hTERT promoter is both strong enough and specific enough to be
used in targeting transgene expression to tumors.
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. The failure of the hTERT promoter to
drive detectable LacZ expression in adult mouse tissues was not
attributable to the inability of the hTERT promoter to use the mouse
transcriptional machinery, inasmuch as a high level of transgene
expression was detected in a mouse lung carcinoma cell line (M109)
after infection with Ad/hTERT-LacZ (data not shown). It is noteworthy
that the promoter region of the mouse TERT gene was also
recently cloned; the E-box and two SP1 binding sites in the core
promoter region, which were believed to be critical for their high
expression in cancer cells, were found to be conserved between hTERT
and mouse TERT (22)
. These data together suggest
that the hTERT promoter can be used to prevent transgene expression in
normal liver and spleen cells and to minimize the liver and spleen
toxicity of a therapeutic gene after its systemic delivery.
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To test whether the hTERT promoter can be used to negate the toxic
effects of the Bax gene on normal cells while preserving its
antitumor activity, we constructed a recombinant adenoviral vector
(Ad/hTERT-GV16) by replacing the PGK promoter in Ad/PGK-GV16
with the hTERT promoter. The effects of the Bax gene on
normal and tumor cells when induced by the hTERT promoter compared with
the effects when induced by the PGK promoter were then tested using the
binary adenoviral vector system (Fig. 3A)
. Human lung cancer lines H1299 and A549, NHBE cells, and
NHFB cells were treated with PBS, Ad/CMV-GFP + Ad/PGK-GV16,
Ad/GT-Bax + Ad/CMV-GFP, Ad/GT-Bax + Ad/hTERT-GV16,
or Ad/GT-Bax + Ad/PGK-GV16. The cells were harvested 48 h after the treatment and subjected to fluorescence-activated cell
sorter analysis to determine the fraction of apoptotic cells by
quantifying the sub-G1 population. Induction of
apoptosis in H1299 and A549 cells was comparable after infection with
either Ad/GT-Bax + Ad/hTERT-GV16 or Ad/GT-Bax + Ad/PGK-GV16, which suggests that the hTERT promoter is as strong as the
PGK promoter in inducing Bax gene expression and apoptosis
in tumor cells. In the two normal cell lines (NHBE and NHFB),
however, treatment with Ad/GT-Bax + Ad/PGK-GV16 elicited
substantial apoptosis as well, whereas treatment with Ad/GT-Bax + Ad/hTERT-GV16 elicited no obvious apoptosis. These results
demonstrated that the hTERT promoter can be used to drive
tumor-specific proapoptotic gene expression and apoptosis induction
while negating the toxicity of a proapoptotic gene to normal cells.
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. The results were further supported by Western blot
analysis. Both hTERT and PGK promoters induced strong Bax expression in
A549 cells. In comparison, PGK but not hTERT promoter induced strong
Bax expression in NHFB cells (Fig. 3C)
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Bax Gene Expression Driven by the hTERT Promoter
Suppresses Tumor Growth in Vivo.
To evaluate the possibility of using the hTERT promoter for in
vivo Bax gene therapy, we established H1299 tumors s.c. in nude
mice and treated the tumors with the Bax gene the expression
of which was driven by the hTERT or PGK promoter. After three
sequential intratumoral injections of adenoviral vectors, tumor size
changes were monitored for 3 weeks. Treatment with Ad/GT-Bax + Ad/hTERT-GV16 or Ad/GT-Bax + Ad/PGK-GV16 resulted in
the same levels of tumor-growth suppression that were significantly
different from treatments with PBS, Ad/E1- or
Ad/GT-LacZ + Ad/hTERT-GV16 groups (P 0.001; Fig. 4
). These results demonstrated that the hTERT promoter can effectively
drive transgene expression in tumors in vivo.
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, panel d, the majority of hepatocytes have
undergone apoptosis after PGK promoter-induced Bax expression. In
comparison, when Ad/GT-Bax + Ad/hTERT-GV16 was infused, very
few apoptotic hepatocytes were observed (Fig. 5A
,
panel c). To further document the liver toxicity by the
Bax gene treatment, blood samples were collected 48 h
after i.v. virus injection, and the serum levels of liver AST and ALT
were determined (Fig. 5B)
. Although treatments with PBS,
Ad/GT-LacZ + Ad/PGK-GV16, or Ad/GT-Bax + Ad/hTERT-GV16 resulted in the same serum AST and ALT levels, the
treatment with Ad/GT-Bax + Ad/PGK-GV16 resulted in >16- and
41-fold increases in AST and ALT levels, respectively
(P 0.0001). Together, these results
suggest that hTERT promoter can be used to prevent the liver toxicity
of proapoptotic genes.
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Discussion |
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Targeted transgene expression may also be achieved by receptor-mediated gene targeting. This method has been exploited primarily for nonviral gene delivery through the use of molecular conjugates and protein/DNA complexes. A variety of ligands and receptors have been used for the delivery of genes to specific tissues or cells, such as targeting hepatocytes via asialoglycoprotein receptor (24) and targeting macrophages via mannose receptors (25) . Targeted gene delivery via viral vectors has also been tested by conjugating preformed vector particles with antibodies, and by manipulating the genes encoding the viral capsid or coat proteins so as to produce viral particles with modified surface proteins. Modification of adenoviral surface proteins to alter the natural tropism of adenovirus and to permit gene transfer into specific cell types has been reported (26 , 27) . However, no tumor-specific gene delivery system with broad application has yet been developed.
We have shown here that the hTERT promoter has high transcriptional activity in a variety of human cancer cell lines and can be used to prevent the toxic effects of the Bax gene in vitro and in vivo without compromising the antitumor activity of the gene. Thus, hTERT should be useful for targeting the pharmaceutical effects of a therapeutic gene to cancer cells. Indeed, the fact that telomerase is active in various tumor types and in >85% of all primary tumors suggests that the hTERT promoter will find broad applications in cancer gene therapy.
It is intriguing that the relatively short hTERT core promoter acts so remarkably differently in tumor versus normal cells, although the mechanisms responsible are still not quite understood. However, the widespread expression of hTERT in heterogeneous tumors suggests that general transcriptional factors, rather than any specific transcriptional factors, are responsible for the up-regulation of hTERT in tumor cells. Several reports have shown that c-Myc, an E-Box binding protein, can directly activate hTERT transcription (16 , 17 , 28 , 29) . On the other hand, although hTERT expression can be activated by c-Myc in telomerase-competent cells, the levels are not as high as those seen in tumor cells, which strongly suggests that active repression mechanisms may exist in normal cells.
One of the major concerns about the use of the hTERT promoter to drive expression of proapoptotic or cytotoxic genes is its potential toxicity to stem cells. However, evidence suggests that hTERT may not be active in quiescent or G0 stem cells (30) . In fact, a study on telomerase activities in bone marrow cells showed that primitive stem cells and their mature derivatives had lower basal levels of telomerase activity, whereas the early progenitors had higher levels (31) , which suggested that toxic effects, if any, on stem cells arising from the hTERT promoter may be transient. Furthermore, the toxic effects on stem cells can also be prevented by selecting vectors that poorly transduce stem cells. For example, adenoviral vectors infect stem cells poorly. A very high dose of an adenoviral vector and prolonged cell-vector contact are required to infect stem cells, and, even at high doses of adenoviral vector, only a limited percentage of stem cells will be infected (32) . Nevertheless, it will be important to investigate the long-term toxic effects of proapoptotic or cytotoxic genes the expression of which is driven by the hTERT promoter delivered by a particular vector system.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from an Institutional
Development Award to the Human Cancer Gene Prevention and Therapy
Program, the W. M. Keck Center for Cancer Gene Therapy; a
research project grant from the American Cancer Society
(RPG-00-274-01-MGO); NIH program project Grant CA78778-01A1; a NIH
grant for a Specialized Program of Research Excellence (SPORE) in Lung
Cancer (P50-CA70907); NIH Core Grant CA 16672 for Medium and Vectors;
and Tobacco Settlement Fund. J. G. is an M. D. Anderson Odyssey
fellow. 
2 To whom requests for reprints should be
addressed, at Department of Thoracic and Cardiovascular Surgery, Box
109, The University of Texas M. D. Anderson Cancer Center, 1515
Holcombe Boulevard, Houston, TX 77030. Phone: (713) 794-4039; Fax:
(713) 794-4669; E-mail: bfang{at}notes.mdacc.tmc.edu
3 The abbreviations used are: CMV,
cytomegalovirus; TERT, telomerase reverse transcriptase; hTERT, human
TERT; NHFB, normal human fibroblast; NHBE, normal human bronchial
epithelial; MOI, multiplicity of infection; X-Gal,
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; XTT,
2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide
inner salt; AST, aspartate transaminase; ALT, alanine transaminase;
PGK, 3-phosphoglycerate.
Received 5/24/00. Accepted 8/16/00.
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, PBS; 
, Ad/CMV-GFP + Ad/PGK-GV16; 
, Ad/GT-Bax + Ad/CMV-GFP; 
, Ad/GT-Bax + Ad/hTERT-GV16; *,
Ad/GT-Bax + Ad/PGK-GV16. C, induction of
Bax gene expression in A549 (Lanes 1–4)
and NHFB (Lanes 5–8) cells after treatment with PBS
(Lanes 1 and 5), Ad/GT-Bax + Ad/CMV-GFP (Lanes 2 and 6),
Ad/GT-Bax + Ad/hTERT-GV16 (Lanes 3 and
7), and Ad/GT-Bax + Ad/PGK-GV16 (Lanes
4 and 8). Western blot analysis was performed
48 h after treatments. Under each lane, the
relative Bax:ß-actin ratio with the PBS-treated cells normalized to
1. Similar results were obtained with H1299 and NHBE cells (data not
shown).
